High cytotoxic activity against herpes simplex virus type 1 infected targets found in murine peritoneum

Unelicited murine peritoneal cells (PC) were found to efficiently lyse the natural cytotoxic (NC) cell target, WEHI-164, as well as herpes simplex virus-type 1 (HSV-1)-infected WEHI-164 and 3T3 cells but not the natural killer (NK) target, YAC-1. Lysis by PC of HSV-1-infected WEHI-164 and 3T3 cells required longer culture times than splenic cell lysis of YAC-1 cells. The PCs which lysed these targets were found to be slightly adherent to nylon wool but non-phagocytic, and were not augmented by preincubation with interferon. Also, PC effectors lacked Qa-5 and asialo GM1 markers which are found on splenic NK cells which lysed YAC-1 targets. We found that there was no correlation between peritoneal NC activity and genetic resistance to HSV-1.     

Natural cytotoxic activity in a cloned natural killer cell line is mediated by tumor necrosis factor

The interleukin-2-dependent mouse natural killer (NK) cell line NKB61A2 concomitantly exhibits NK and natural cytotoxic (NC) activities. This was determined by the cells' ability to lyse both the NK-sensitive YAC-1 lymphoma and the NC-sensitive WEHI-164 fibrosarcoma cell lines in a 4- and 18-hour 51Cr release assay, respectively. Cell-free supernatant from NKB61A2 cells grown in culture for 48 h had substantial lytic activity against WEHI-164. The mouse mast cell line PT18-A17 and the rat basophilic leukemia cell line RBL-2H3, which both express NC activity, also produced a soluble factor during culture which lysed WEHI-164 cells. This activity was increased in the basophilic/mast cells by crossbridging the surface IgE receptors. Similar results were obtained by triggering the basophilic NC cells with the calcium ionophore ionomycin and the tumor promoter phorbol-12-myristate-13-acetate (PMA). Such triggering of NKB61A2 cells, however, did not significantly increase their NC activity. Interestingly, both ionomycin and PMA had an inhibitory effect on the NK activity of NKB61A2. Recently it has been found that tumor necrosis factor (TNF) is a major mediator of NC activity. To determine if the soluble factor responsible for the NC activity of the NK clone was related to TNF, a rabbit polyclonal antiserum to mouse TNF was tested against the cell-free culture medium of NKB61A2, PT18-A17, RBL-2H3 and murine recombinant TNF (Mu-rTNF). The lytic activity of the culture medium from all these cells and the Mu-rTNF control was abrogated by this antibody. These data suggest that the murine cell line NKB61A2 has both NK and NC activities and that the NC activity is due to a factor immunologically similar to TNF. In addition, the enhancement of NC activity in the NK cell line is apparently under control by a separate pathway, different from that in the basophilic cells.     

Tumor necrosis factor-mediated cytotoxicity by tumor-associated macrophages

We have directly demonstrated that macrophages present within solid EMT6 mammary tumors (of BALB/c origin) produce TNF-alpha (TNF). These tumor-associated macrophages lysed WEHI-164, a TNF-sensitive cell line, very efficiently. This cytotoxicity was abrogated in the presence of anti-TNF antisera. In contrast, EMT6 cells, the tumor from which the macrophages were obtained, were not effectively lysed by the macrophages and were 100-fold less sensitive to lysis by recombinant mouse TNF. Thus, marked heterogeneity exists among tumors regarding sensitivity to TNF-mediated cytotoxicity. Similarly, macrophages which infiltrate into EMT6 multicellular spheroids implanted into the peritoneal cavity as well as free cells within the cavity exhibited TNF-mediated cytotoxicity of WEHI-164 cells, but failed to lyse EMT6 cells. The kinetics of lysis by these cells was similar to that of recombinant mouse TNF.     

Cell-associated tumor necrosis factor (TNF) as a killing mechanism of activated cytotoxic macrophages

Different macrophage populations were investigated for their abilities to secrete tumor necrosis factor (TNF) and to lyse TNF-susceptible tumor cells. In this way we could demonstrate that TNF-secretion, although a feature of all activated macrophage populations, is no absolute requirement for the killing of the TNF-sensitive Wehi 164 target. Macrophage cytotoxicity against this cell but not against the TNF-resistant P815 mastocytoma, was completely inhibitable by a specific anti-TNF serum also in the absence of measurable secreted TNF. Moreover the TNF-dependent lysis of tumor cells could also be performed by activated macrophages that had been fixed with paraformaldehyde before the addition of the target cells. In the indirect radioimmunoassay, TNF could be demonstrated on the surface of fixed effector cells. Our results must be interpreted in terms of membrane-associated TNF as the lytic principle for TNF-susceptible tumor cells.     

Morphology and lytic mechanisms of interleukin 3-dependent natural cytotoxic cells: tumor necrosis factor as a possible mediator

Populations of interleukin 3 (IL 3)-dependent cells can be derived from mouse bone marrow that display natural cytotoxicity (NC) against Wehi-164 target cells but do not display natural killing against YAC-1 cells. These bone marrow-derived NC cells cultured up to 2 mo in IL 3 do not contain rearranged T cell receptor beta-chain genes. They appear to be mast-like cells by electron microscopy and contain heterogeneous type granules. The molecules that mediate NC appear to be contained in these granules and are preformed because protein synthesis inhibitors have no effect on the capacity of IL 3-dependent NC cells to lyse Wehi-164 target cells. In addition to the IL 3-dependent bone marrow-derived cells, the basophilic leukemia cells, RBL-1, but not P815 mastocytoma cells were found to mediate NC against Wehi-164 cells. Both bone marrow-derived NC and RBL-1 cells can lyse L929 cells in 18 hr, suggesting that the putative NC mediator may be related to lymphotoxin/tumor necrosis factor (TNF). Recombinant human TNF displayed identical properties as NC cells; both entities possessed the same target cell specificity and had similar kinetics of target cell killing. The use of polyclonal rabbit antimouse TNF antibody blocked the actions of NC cells. Thus we believe that the mediation of NC is through the actions of a TNF-like molecule.     

Natural cytotoxic cell-specific cytotoxic factor produced by IL-3-dependent basophilic/mast cells. Relationship to TNF

The murine IL-3-dependent mast cell line, PT18-A17, and the rat basophilic leukemia cell line, RBL-2H3, were found to mediate natural cytotoxic (NC) activity via the release of a soluble factor which specifically lysed NC-sensitive WEHI-164 but not NK-sensitive YAC-1 tumor cells. The release of this NC cell-specific cytotoxic factor was enhanced by triggering of both types of cells via IgE receptor bridging. This factor had activity on TNF-sensitive but not TNF-resistant cell lines and could be neutralized by two independently produced polyclonal anti-mouse TNF antisera. It was not neutralized by antibodies against mouse IFN-alpha/beta or IFN-gamma. Moreover, it was not neutralized by a monoclonal or a polyclonal anti-human TNF, demonstrating that the rodent TNF differed antigenically from human TNF. These results indicate that the cytotoxic factor released from a murine IL-3-dependent mast cell line and from a rat basophilic leukemia cell line is immunologically and functionally related to murine TNF.     

Hemorrhage induces enhanced Kupffer cell cytotoxicity while decreasing peritoneal or splenic macrophage capacity. Involvement of cell-associated tumor necrosis factor and reactive nitrogen.

Studies indicate that simple hemorrhage produces a profound depression of cell-mediated immunity, thereby contributing to an enhanced susceptibility to septic challenge in the host. However, it remains unknown whether or not the macrophages' cytotoxic capacity is altered after hemorrhage. To study this, C3H/HeN mice were bled to and maintained at a blood pressure of 35 mm Hg for 60 min, and adequately resuscitated. Mice were then killed at 2 or 24 h after hemorrhage to obtain peritoneal macrophage, splenic macrophage, and Kupffer cells. Cytotoxicity was assessed by determining the capacity of these macrophages to lyse [3H]TdR labeled WEHI-164 clone 13 or P815 tumor target cells (WEHI-164, sensitive to both soluble and cell-associated TNF vs P815 cells, insensitive to soluble TNF). Peritoneal and splenic macrophages from hemorrhaged animals exhibited a significantly reduced cytotoxic capacity, whereas Kupffer cells' ability to kill the target cells was enhanced. Similarly, the Kupffer cells' capacity to release TNF and IL-1, as well as express cell-associated forms of this cytokine are significantly enhanced on macrophages isolated 2 h after hemorrhage, whereas peritoneal macrophages are not. Furthermore, antibodies directed at mouse TNF but not against murine IL-1 alpha or murine IL-6 were able to oblate the enhanced target cell lysis of unfixed, as well as paraformaldehyde fixed (metabolically inactive) Kupffer cells. Studies using inhibitors (GN-monomethyl-arginine, superoxide dismutase, catalase, and ibuprofen) of other TNF-inducible mechanisms of target cell killing indicated that only the inhibition of the release of reactive nitrogen consistently depressed the cytotoxic capacity of Kupffer cells from hemorrhaged mice. Thus, the increased Kupffer cell cytotoxicity from hemorrhaged mice is most likely mediated through the expression of cell-associated TNF and the release of reactive nitrogen.     

Enhanced lysis of herpes simplex virus type 1-infected mouse cell lines by NC and NK effectors

Spontaneously cytotoxic murine lymphocytes lysed certain cell types infected by herpes simplex virus type 1 (HSV-1) better than uninfected cells. The levels of virus-directed lysis varied widely from target to target, and we found that differences in virus-directed lytic efficiency could be attributed both to the characteristics of HSV-1 replication in the different targets and to the subgroup of natural effector cells which mediated lysis. Although HSV-1 adsorbed to the surface of all the target cells, those in which the virus replicated more efficiently were lysed to a greater extent. As targets, we used cell lines that, when uninfected, were spontaneously lysed by NK cells (YAC-1) or by NC cells (WEHI-164). We also used a fibroblastoid cell line (M50) and a monocytic tumor line (PU51R), which were not spontaneously killed. Using complement-mediated elimination of Qa-5-positive or asialo-GM1-positive NK cells to distinguish NK from NC activity, we found that NK cells lysed HSV-1-infected YAC cells better than uninfected cells, and an NC-like activity selectively lysed HSV-1-infected WEHI cells. In addition, we showed that both NK and NC cytotoxicities contributed to the lysis against the HSV-1-infected fibroblastoid line, M50, but the infected PU51R cells were killed by only NK effectors. These findings were consistent with the results of experiments performed to define the role of interferon in induction of virus-augmented cytolysis. Increased lysis of YAC-HSV and PU51R-HSV was entirely due to interferon activation and was completely abolished by performing the 51Cr-release assay in the presence of anti-interferon serum. Because NC activity was not augmented by interferon, virus-enhanced NC lysis of M50-HSV and WEHI-HSV was not due to this nonspecific mechanism. Together, our data show that HSV-1 infection of NK/NC targets induces increased cytotoxicity, but the effector cell responsible for lysis is determined by the uninfected target, or by an interaction between the virus and target cell, rather than by a viral determinant alone.     

Ultraviolet light-induced increase in tumor cell susceptibility to TNF-dependent and TNF-independent natural cell-mediated cytotoxicity

Previously we demonstrated that two consecutive in vitro irradiations of MCA 102 cells with high doses of UVC light (610 and 457 J/m2) resulted in a selection of a permanent line MCA 102UV that manifested high sensitivity to natural cell-mediated cytotoxicity (NCMC). In the present study analysis of the effector cells involved in lysis of these tumor cells was performed by comparing the cytotoxicity of normal spleen cells which mediated both NK and NC cell activity with (a) normal spleen cells in which NC activity was neutralized by anti-TNF Abs (NK+,NC-), (b) NK-depleted or NK-deficient spleen cells (NK-,NC+), and (c) NK-deficient or -depleted spleen cells with NC activity neutralized by anti-TNF Abs (NK-,NC-). Results of these studies indicate that lysis of the original MCA 102 tumor cells was relatively low and was mediated by NC cells. UV irradiation significantly increased MCA 102 tumor cell sensitivity to lysis by both NK and NC cells. Analysis of the mechanisms involved in UV-induced NK sensitivity revealed that UV irradiation increased tumor cell susceptibility to lytic NK-derived granules. NC sensitivity of MCA 102UV tumor cells was associated with their increase in sensitivity to TNF and selection of MCA 102UV cells for resistance to rTNF resulted in a decrease in their susceptibility to NC cells. To determine how fast UV-induced sensitivity to NCMC and rTNF can be established, 51Cr-labeled MCA 102 cells were irradiated in vitro with 38-304 J/m2 of UVC light and their sensitivity to lysis by spleen cells and rTNF was tested immediately in an 18-hr cytotoxicity assay. UV treatment with the same doses was repeated 12 days later. The data obtained showed that tumor cell sensitivity to NCMC and TNF appeared shortly after UV irradiation, was stable, and was further substantially augmented by the second round of UV treatment. Thus, in vitro UV irradiation of tumor cells could be an effective modulator of tumor cell sensitivity to TNF-dependent and TNF-independent cell-mediated cytotoxicity.     

Differences in the tumor necrosis factor-alpha-mediated lysis by fixed natural cytotoxic cells and fixed cytotoxic macrophages

TNF-alpha has been shown to be associated with macrophage cell membranes in such a way as to retain cytolytic activity despite fixation of the macrophage effector cells with paraformaldehyde. In this paper we report that, similar to cytotoxic macrophages, natural cytotoxic (NC) cells also use cell-associated TNF to lyse sensitive target cells. However, in contrast to fixed cytotoxic macrophages, NC cells do not retain cytolytic activity after fixation with paraformaldehyde. Additionally, the cytolytic activity of paraformaldehyde-fixed NC cells is not increased by incubation with LPS or by incubation with rTNF before fixation. Western blot analysis indicates that, unlike macrophages, NC cells use a smaller (17 kDa) constitutively active form of TNF. These results indicate that, although both macrophages and NC cells use effector cell-associated TNF to mediate lysis of sensitive targets, the way in which TNF is associated with these two types of effector cells must be different.     

Tumor growth in vivo selects for resistance to tumor necrosis factor

The relationship between in vivo tumor growth and resistance to TNF in WEHI-164 cells has been examined. When a highly TNF-sensitive clone of WEHI-164 was grown in vivo in syngeneic mice it became resistant to rTNF such that a 4 to 5 log higher concentration of TNF was required to produce tumor lysis in vitro. When compared with an in vitro selected TNF-resistant variant, the in vivo selected line was significantly more tumorigenic. The resistant phenotype of both the in vivo and in vitro selected variants was stable in culture and both selected lines were also resistant to lysis by syngeneic spleen cells with natural cytotoxic activity. The parental clone and the two variants were equally sensitive to lysis by allo-CTL and expressed similar levels of MHC class I Ag. Resistance to TNF in the two variants was not a function of de novo production of TNF measured as supernatant TNF activity or TNF mRNA expression. These studies are the first to demonstrate that in vivo tumor growth results in resistance to TNF and therefore may have direct relevance to the efficacy of TNF in the treatment of human neoplasms.     

Natural cytotoxic cells and tumor necrosis factor activate similar lytic mechanisms

The lytic activity of natural cytotoxic (NC) cells has several characteristics which clearly distinguish it from other cell-mediated lytic activities and from most soluble cytolytic factors. An exception is the lytic activity mediated by tumor necrosis factor (TNF). In this paper, we report a detailed comparison of NC and TNF lysis of target cells which are used as prototype NC targets or TNF targets, and show that the two cytolytic activities have very similar, if not identical, lytic mechanisms. We present data showing that target cells which are NC-sensitive are also TNF-sensitive and that target cells which are NC-resistant are also TNF-resistant. Moreover, cells selected either in vivo or in vitro for NC resistance are selected for TNF resistance, and cells selected for TNF resistance are selected for NC resistance. The analysis of the kinetics of 51Cr release mediated by NC cells or by TNF show that both activities affect similar kinetics, in that there is no cell lysis for several hours after targets and effectors first interact. However, NC and TNF lytic activities can be distinguished. By using the cell lines 10ME or B/C-N as targets, it can be shown that whereas NC-mediated lysis is dependent on protein synthesis, TNF-mediated lysis is not. We also show that targets which are resistant to NC-mediated lysis because they express a protein synthesis-dependent resistance mechanism also require protein synthesis to resist TNF-mediated lysis, suggesting that the same resistance mechanism protects cells against both NC cells and TNF. Together, these data strongly support the hypothesis that NC cells and TNF activate the same lytic mechanism within target cells and that TNF may mediate the lytic activity of NC effector cells.     

Cell lines cultured at high density are resistant to lysis by tumor necrosis factor and natural cytotoxic cells

It has been suggested that natural cytotoxic (NC) cell activity and tumor necrosis factor (TNF), the molecular mediator of NC activity, are capable of protecting individuals against the progression of incipient tumors or could be useful in cancer therapy regimens. Much of this speculation arises as a result of in vitro studies, on a variety of tumor cells, demonstrating the cytolytic and cytostatic properties of NC and TNF activities. Here, evidence is presented showing that certain mouse fibroblast cell lines, generally considered sensitive to NC and TNF lysis, are quite resistant to these lytic activities when cultured at high cell density. Although a soluble factor that renders these same target cells resistant to NC and TNF lysis has been described, no such factor is involved in this high density-induced resistance. Rather, it appears that cell to cell contact of the targets is critical. Moreover, the induced resistance to NC and TNF lysis does not result from loss of either NC recognition determinants or TNF receptors by the target cells, but is the consequence of increased expression of a protein synthesis-dependent resistance mechanism. These observations raise the issue of the in vivo phenotype of cells characterized in vitro as sensitive to NC and TNF lysis. It is entirely possible that certain cells which are considered sensitive to NC and TNF activities are, in fact, resistant to these cytolytic activities when growing as tumors (i.e., at high cell density). Should this be the so, NC and TNF cytolytic activities may not function in vivo or may function only via some indirect means.     

Improvement of potential therapeutic value of tumor necrosis-alpha (TNF-alpha) by charge modulation in the tip region

Analysis of published data reveals that the introduction of more basic amino acid residues in the flexible N-terminal region of the human tumour necrosis factor alpha (TNF) molecule indicates a weak but consistent trend towards increased in vitro cytotoxicity, especially when the effect of N-terminal length is taken into account. In our laboratory, a series of TNF analogues with a charge modification in the tip region of the molecule was prepared, and cytotoxicity measured. Similar trends in cytotoxicity with increasing basicity of the TNF analogue were found in this study for two mouse cell lines, L929 and WEHI-164 clone 13-1, as well as for the human line KYM-1D4. For the series of analogues as a whole, a general increase in in vitro cytotoxicity with increasing pI values was not apparent, but some analogues with charge reversal in the tip region, for example, the LK-805 analogue (E107K), exhibited significantly increased cytotoxicity in comparison to native TNF in a range of cell lines, including L929, KYM-1D4-K, WEHI-164 clone 13-1, HEPA 1-6 and EAhy926 cell lines. Experiments with heparinase-pre-treated cells demonstrated that the increased in vitro cytotoxicity of LK-805 is most probably due to interactions with cell surface heparan sulphates that effectively concentrate it before binding to TNF receptors occurs. Examination of structural models of TNF bound to soluble TNF receptor 1 (TNFR1) indicates that simple mutations in the tip region most probably cannot interact with receptor binding sites, and therefore do not directly modulate cytotoxicity.     

Calcium-dependent natural killer and calcium-independent natural cytotoxic activities in an IL-2-dependent killer cell line

Using a cloned murine cell line, NKB61A2, that concomitantly exhibits both NK and natural cytotoxic (NC) activities, we investigated the biochemical mechanisms involved in natural cell mediated cytotoxicity against NK-sensitive YAC-1 tumor cells and against the NC-sensitive WEHI-164 tumor cells. Recent reports have suggested that target cell lysis by cytotoxic lymphocytes occurs by either a calcium dependent and/or a calcium-independent mechanism(s). To determine the role of calcium in NK and NC activities of the NKB61A2 cell line, we evaluated the effect of: 1) extracellular Ca2+ depletion by the divalent cation chelator, EGTA, 2) Ca2+ influx blockade by the Ca2+ channel blocker verapamil, and 3) blocking of intracellular Ca2+ mobilization by 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8). We found that EGTA, verapamil, and TMB-8 were all capable of inhibiting NK activity, but they had little effect on NC activity of the NKB61A2 cells. Using 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide which are inhibitors of protein kinase C and calmodulin respectively, we determined that protein kinase C and calmodulin do play a role in the NK activity of NKB61A2 cells. 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and N-(6-aminohexyl)-5-chloro-1-naphthalanesulfonamide, similar to Verapamil and TMB-8, had no effect on NC activity. Thus, the data indicate that the NK activity of NKB61A2 cells is calcium dependent whereas NC activity is not. These results may explain the disparate reports seen in the literature of calcium-dependent and -independent lysis of tumor cells.     

The cloned cell line L10A2.J expresses natural cytotoxic activity

The analysis of natural cytotoxicity (NC) has been hampered by the lack of cloned NC effectors. In studies reported here we show that the cloned cell line L10A2.J expresses properties similar to those of splenic NC effectors. L10A2.J cells lyse NC-sensitive targets, but do not lyse NC-resistant targets which are sensitive to lysis by natural killer (NK) or cytotoxic T lymphocytes. The mechanism by which L10A2.J cells lyse NC-sensitive targets is similar to the lytic mechanism of splenic NC effectors in that both result in the release of 51Cr from targets with a lag of 5-7 hr after effectors and targets are mixed. In addition, inhibition of protein synthesis during the in vitro assays of NC or L10A2.J lytic activity causes some NC-resistant targets to become sensitive to lysis by both NC and L10A2.J effectors. The only functional difference detected between L10A2.J and splenic NC effectors is in their recognition of targets. While L10A2.J and splenic NC effectors recognize many of the same targets (NC resistant and NC sensitive), L10A2.J, unlike splenic NC effectors, does not recognize the NK-sensitive cell line YAC-1.     

Alveolar macrophage cytotoxic activity is inhibited by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a carcinogenic component of cigarette smoke

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a carcinogenic compound of cigarette smoke that generates electrophilic intermediates capable of damaging DNA. Recently, we have shown that NNK can modulate mediator production by alveolar macrophages (AM) and bronchial and alveolar epithelial cells, suggesting that cigarette smoke can alter lung immune response. Thus, we investigated the effect of NNK and cigarette smoke extract (CSE) on AM capacity to eliminate tumoral cells. Rat AM cell line, NR8383, was treated with NNK (500 μM) or CSE (3%) and stimulated with lipopolysaccharide (10 ng/ml). The release of cytotoxic mediators, tumor necrosis factor (TNF) and reactive oxygen species (ROS), was measured in cell-free supernatants using ELISA and superoxide anion production. TNF- and ROS-dependent cytotoxicity were studied using a ⁵¹Chromium-release assay and WEHI-164 and P-815 cell lines. Treatment of AM with NNK and CSE for 18 h significantly inhibited AM TNF release. CSE exposure resulted in a significant increase of ROS production, whereas NNK did not. TNF-dependent cytotoxic activity of NR8383 and freshly isolated rat AM was significantly inhibited after treatment with NNK and CSE. Interestingly, although ROS production was stimulated by CSE and not affected by NNK, CSE inhibited AM ROS-dependent cytotoxicity. These results suggest that NNK may be one of the cigarette smoke components responsible for the reduction of pulmonary cytotoxicity. Thus, NNK may have a double pro-carcinogenic effect by contributing to DNA adduct formation and inhibiting AM cytotoxicity against tumoral cells.     

Cellular models of macrophage tumoricidal effector mechanisms in vitro. Characterization of cytolytic responses to tumor necrosis factor and nitric oxide pathways in vitro

The recently described L-arginine-dependent nitric oxide (NO) pathway has been proposed to interact synergistically with the TNF pathway in murine macrophage-mediated tumor cytotoxicity in vitro. We have employed an experimental construct in which these two pathways were independently expressed by two different effector cell populations. The TNF-dependent pathway was committed by murine 3T3 cells transfected with the cDNA encoding human pro-TNF. The NO pathway was executed by the murine EMT-6 mammary adenocarcinoma cell line treated with murine rIFN-gamma and LPS. Controls for the TNF pathway committed by the transfectant included lysis of the TNF-sensitive murine L929 cell in coculture, secretion of TNF, and absence of nitrite synthesis. For the NO pathway controls included lysis of the murine P815 mastocytoma cocultured with activated EMT-6 cells that had been pretreated with murine rIFN-gamma and LPS, production of nitrite by this activated effector cell, and an absence of TNF secretion. The target cell panel included L929, EMT-6, P815, and murine B16 melanoma and TU-5 sarcoma cell lines. All targets on this panel were susceptible to lysis by LPS-triggered murine bacillus Calmette-Guérin-activated macrophages. The 3T3 transfectant caused significant lysis of cocultured L929 and TU-5 targets. The EMT-6 effector cell only caused significant lysis of the P815 target. When both effector cells were cocultured with these target cells, lysis of the P815 target was observed to be additive or superadditive; however, for all the other targets, cytotoxicity was comparable with or subadditive compared with that seen with the 3T3 transfectant effector cell alone. Thus, these two pathways do not appear to account for the broad, potent tumoricidal activity observed for activated macrophages in vitro.     

The adenovirus E3-14.7K protein is a general inhibitor of tumor necrosis factor-mediated cytolysis

We have previously described a 14,700 m.w. protein (14.7K) encoded by the E3 region of adenovirus that prevents TNF-mediated cytolysis of adenovirus-infected C3HA mouse fibroblasts. In the studies described here we have extended our analysis of TNF cytolysis of C3HA cells and the circumstances under which 14.7K protects these cells from cytolysis. C3HA cells were killed by TNF in the presence of inhibitors of protein synthesis, in the presence of cytochalasin E (which disrupts the microfilaments), and when adenovirus E1A was expressed. As described for other cell types, pretreatment of C3HA cells with TNF prevented cytolysis by TNF plus cycloheximide or TNF plus cytochalasin E, indicating that TNF induces a response that protects against these treatments. Remarkably, when 14.7K was expressed in virus-infected cells, it also prevented TNF-induced lysis whether sensitivity to TNF was induced by inhibition of protein synthesis, disruption of the cytoskeleton by cytochalasin E, or expression of adenovirus E1A. The 14.7K protein also prevented TNF lysis of cells that are spontaneously sensitive to TNF lysis. Thus, 14.7K appears to be a general inhibitor of TNF cytolysis, and as such should be an important tool in unraveling the mechanism of TNF cytolysis. There was one exception; NCTC-929 cells were spontaneously sensitive to TNF lysis and that lysis was not affected by 14.7K even though the protein was made in large quantities and was metabolically stable in these cells. This suggests that there is heterogeneity among TNF-sensitive cell lines. The 14.7K protein was found in both the nuclear and cytosol fractions of TNF resistant as well as all spontaneously sensitive cells suggesting that 14.7K may have more than one site of action within the cell.